JAC
Journal of Antimicrobial Chemotherapy (2000) 46, 297–301
Antimicrobial resistance of mesophilic Aeromonas spp. isolated from two European rivers Marisol Goñi-Urrizaa, Lionel Pineaub, Michèle Capdepuya, Christine Roquesb, Pierre Caumettec and Claudine Quentina* a
Laboratoire de Microbiologie, Faculté de Pharmacie, Université de Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex; bLaboratoire de Microbiologie, Université de Toulouse 3, 31–35 rue des Maraîchers, 31400 Toulouse cedex; cLaboratoire d’Ecologie Moléculaire, Université de Pau, BP 1155.F, 64013 Pau Cedex, France The activity of 19 antibiotics and four antiseptics and/or disinfectants was studied against 138 non-redundant strains of Aeromonas spp. (104 Aeromonas caviae, 22 Aeromonas sobria and 12 Aeromonas hydrophila) isolated from two European rivers. Antibiotic resistance frequencies were: nalidixic acid, 59%; tetracycline, 14%; fosfomycin, 8%; tobramycin and cotrimoxazole, 7%; cefotaxime, 4%; chloramphenicol, 2%; gentamicin, 1%. Most of the nalidixic acid-resistant strains were susceptible to fluoroquinolones (54–98%). Antibiotic resistance rates varied according to the source of the strains. All Aeromonas spp. strains were killed by 50 ppm of chlorine, cetylpyridinium chloride and peracetic acid, and by 1600 ppm of glutaraldehyde.
Introduction
Materials and methods
Mesophilic, motile Aeromonas spp. are normal inhabitants of soil and freshwater. Over the last three decades, these organisms have also emerged as opportunistic pathogens responsible for gastroenteritis, skin and soft tissue infections and a variety of clinical syndromes in compromised patients.1 Antibiotic sensitivity of clinical isolates of Aeromonas spp. has been extensively studied,2–4 but less is known about environmental strains;4–5 antiseptic and disinfectant susceptibility of the genus has received little attention, except for chlorine.6 However, fluvial waters receive human and animal wastewater discharges, which are expected to contain antimicrobial agents likely to exert a selective pressure, and commensal resistant bacteria, capable of transferring their resistances to autochthonous bacteria. Consequently, the freshwater indigenous flora may become a reservoir for antimicrobial resistance genes, and the reuse of these waters for humans and animals may contribute to the limitation of antimicrobials’ efficiency. The aim of this study was to determine the antimicrobial resistance rates among riverine Aeromonas spp., taken as representatives of the autochthonous flora, in order to evaluate the level of antibacterial resistance in polluted river sites and the potential risk that it represents.
Water samples (500 mL) were collected during 10 sampling campaigns at 16 sites scattered along 30 km either of the Arga (Spain) or the Garonne river (France), near urban effluent discharges (Pamplona and Bordeaux, respectively), in 1996. The number of Gram-negative bacteria varied from 1.5 102 to 6.5 105 per mL according to the sampling site and date. Aeromonas strains were identified to the phenospecies level according to standard criteria.4 Antibiograms were performed by the disc diffusion method, using 22 antibiotics. After elimination of the redundant strains (i.e. strains collected from the same site at the same moment exhibiting the same antibiotype), 138 strains were examined. MICs of antibiotics were determined by a standard agar dilution method using Mueller–Hinton medium.7 MBCs of antiseptics and/or disinfectants were determined by a dilution-neutralization micromethod.8
Results and discussion Strains of the Aeromonas caviae complex (104 strains, 75%) were much more abundant than those of the Aeromonas sobria (22 strains, 16%) and Aeromonas hydrophila (12
*Corresponding author. Tel: 33-5-57-57-10-75; Fax: 33-5-56-90-90-72; E-mail:
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297 © 2000 The British Society for Antimicrobial Chemotherapy
M. Goñi-Urriza et al. strains, 9%) complexes. All three groups have been recovered from human infections, although A. hydrophila and A. sobria appear to be inherently more invasive and pathogenic than A. caviae.1 The MICs of 19 antibiotics were determined for all strains (Table I). The intrinsic susceptibility of Aeromonas spp., as judged by the MIC50, was quite similar to that previously reported.1,3–5 Indeed, Aeromonas spp. are known to be intrinsically susceptible to all antibiotics active against non-fastidious Gram-negative bacilli, except for many β-lactams, due to the production of multiple inducible, chromosomally encoded β-lactamases.1,9 In this study, most Aeromonas spp. strains were resistant to ampicillin (99%), ticarcillin (87%) and cephalothin (93%); 56% were cefoxitin resistant. Resistance to third-generation cephalosporins and imipenem is known to be associated with the derepression of the chromosomal enzymes.1,9 Among the strains studied, 4% gave cefotaxime MICs of 8–64 mg/L which is a similar rate to that reported in earlier studies (0–3%),4,5 but much lower than recently observed by Ko et al. (19%).3 No imipenem-resistant strains were encountered, although four strains of A. sobria and one A. hydrophila gave MICs of 1–4 mg/L, compared with 5–9% elsewhere.3,4 β-Lactam agents should be avoided in the treatment of Aeromonas spp. infections, even if MICs are still in the susceptible range, since resistant mutants overproducing their chromosomal β-lactamases may be selected during therapy.1 Our results confirm that Aeromonas spp. are poorly susceptible to streptomycin (MIC50 16 mg/L) and, to a lesser extent, to kanamycin (MIC50 4 mg/L); gentamicin is slightly more active than tobramycin (MIC50 1 mg/L). Five strains (4%) exhibited a resistance phenotype consistent with the production of desoxystreptamin modifying enzymes, conveying variable levels of resistance to kanamycin (16–512 mg/L), tobramycin (2–64 mg/L) and gentamicin (2–16 mg/L). Aeromonas spp. usually retain their aminoglycoside susceptibility.1 However, tobramycinresistant strains have been reported by Chang & Bolton (0–10%),2 and Ko et al. (25%).3 The latter authors also found as many as 49% tetracycline-resistant Aeromonas spp., compared with 8–22% in the literature,1,2 and 14% here (MIC 8–128 mg/L). Four additional strains were lowlevel resistant (2–4 mg/L). Chloramphenicol resistance is an extremely rare trait in Aeromonas spp.1 Thus, Montoya et al.5 reported a single strain highly resistant to chloramphenicol (MIC 128 mg/L). However, Chang & Bolton2 found 8% of strains resistant to chloramphenicol 10 mg/L. Seven of our strains (5%) exhibited a low-level resistance toward this antibiotic (MIC 8–32 mg/L). Low-level resistance to tetracycline and chloramphenicol in Aeromonas spp. has been shown to result from decreased permeability. Neither sulphamethoxazole nor trimethoprim alone is very active against Aeromonas spp., but co-trimoxazole is generally efficient, due to the strong synergy between the drugs.1 Accordingly, the respective MIC50s were 256, 4 and 4 mg/L. The nine co-trimoxazole-resistant strains (7%, instead of 39–50% for Ko et al.3) exhibited MICs of
sulfamethoxazole 512 mg/L and/or MICs of trimethoprim 64 mg/L. As regards fosfomycin, the strains divided into three populations: highly susceptible (0.2–8 mg/L, 78%), moderately susceptible (16–128 mg/L, 19%) and resistant (512 mg/L, 3%). All strains were inhibited by 4 mg/L of colistin. Surprisingly, most of the strains (59%) were resistant to nalidixic acid (MIC 8 mg/L). The sensitivity of these 81 strains to seven other quinolones was investigated further (Table I). Most of them were resistant to first-generation quinolones (pipemidic acid and oxolinic acid, 67%), but clinically susceptible to fluoroquinolones [from pefloxacin (54%) to ciprofloxacin (98%)], despite increased MICs. Aeromonas spp. strains have been found to be uniformly susceptible to quinolones,2 until recently: Ko et al.3 obtained MICs of fluoroquinolones 4 mg/L. Excluding quinolones, 63% of the strains exhibited a wildtype antibiotic susceptibility pattern. The three Aeromonas complexes shared a similar antibiotic susceptibility pattern, except for a greater imipenem sensitivity of A. caviae, possibly related to the absence of metallo-β-lactamase in this phenospecies.9 Conflicting data in the literature have been explained by the taxonomic composition of the phenospecies, the method of isolation and the source of the strains.1–4 Most of our strains came from the Arga river (100 isolates, compared with 38 from the Garonne river). Antibiotic resistance frequencies and profiles varied according to the source of the strains: nalidixic acid resistance was widespread in both rivers, but represented 72% in the Arga river versus 26% in the Garonne river; the most common resistances to other antibiotics involved trimethoprim (42%) and tetracycline (18%) in the Arga river, but trimethoprim (42%) and fosfomycin (18%) in the Garonne river. Aeromonas isolates are generally susceptible,1,5 but unusually high levels of resistant strains have been reported in Asia.2,3 A higher incidence of antibiotic resistance in environmental Aeromonas spp. is associated with areas of heavy human impact. In this study, most of the antibiotic resistances in riverine Aeromonas spp. are probably related to the presence of humans, and their variation probably reflects local antibiotic usage. The MBCs of four antiseptics and/or disinfectants were determined for the 138 strains of Aeromonas spp. (Table II). A concentration of 50 ppm of free chlorine, peracetic acid and cetylpyridinium chloride was required to kill 100% of the strains (respective MBC50s 12.5, 1.57 and 6.25 ppm). Chlorine is used widely for the decontamination of water distribution systems and in the food industry; however, Aeromonas spp. may be found in chlorinated drinking water supplies, even with low coliform counts.6 Chlorine resistance of Aeromonas spp. must therefore be taken into account, since the main sources of enteric infections associated with these organisms are domestic water supplies.6 Peracetic acid might be a useful alternative agent for water disinfection. Collyria containing cetylpyridinium chloride are expected to be a valuable antiseptic treatment for
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Table I. Antibiotic susceptibility of riverine Aeromonas strains Number of strains with a MIC (mg/L) of: Antibiotic
Pipemidic acid Oxolinic acid Flumequine Pefloxacin Sparfloxacin Norfloxacin Ciprofloxacin a
138 138 138 138 138 138 138 138 138 138 138 138 138 138 138 138 138 138 138 81a 81a 81a 81a 81a 81a 81a 81a
Nalidixic acid-resistant strains.
0.1
70 110
1 1 2 1
0.2
0.5
3 31 14
6 15 9
2 10 30 1
1 18 55 72 22 2
4 17 47 3
2 7 3 27
1
512
2
4
8
16
32
64
128
256
512
1 2 1 6 11 2 1 5 54 36 10 67
4 12 3 2 1 27 37 26 2 36
1 3 1 20 3 1 11 52 17 8 2 4
2 7 3 14 1
6 5 19 2
18 7 2 1
3 16 6 6 1
10 26 12 11
18 31 21 20
29 15 38 14
74 14 40 5
9 5
24 1 1
14 1
9
6 1
4
28 30 46 30 1 16 16
24 9 1 1 6 2 1 22 3 12
18 26 22 23 13 1
2 15 20 15 7
5 30 8 28 22
14 4 29 52 10 31 32
6 3 8 9 22
1 3 14 14 27 18
8 4 25 20 16 12
36 79 22 7 1 1 1 3 27 15 12 7 11 1
35 36 7 1 1 4 18 9 4
24 14 5 2 1
22 6 12 3 1
12 1 3 11 4 7
1 10 2 4 4
24 1 2 3
37 1 1
27 1 1 1
36 2 1 3
1
9
19
31
10
11
10 6 6
3 1 2
16
3
2
MIC breakpoint Resistant (mg/L)7 strains (%) 8 32 16 16 8 8 16 16 8 8 8 16 128 8 80 64 4 16 2 2 16 4 8 2 2 2 2
99 87 93 56 4 0 65 12 7 1 14 2 90 42 7 8 5 59 12 21 67 67 33 46 40 32 2
Antimicrobial resistance of river strains of Aeromonas spp.
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Ampicillin Ticarcillin Cephalothin Cefoxitin Cefotaxime Imipenem Streptomycin Kanamycin Tobramycin Gentamicin Tetracycline Chloramphenicol Sulphamethoxazole Trimethoprim Co-trimoxazole Fosfomycin Colistin Nalidixic acid Ofloxacin
No. of strains
1 1 2 8 39 15 27 10 3 1 39 12
Aeromonas-associated ocular infections. Most Aeromonas spp. strains were killed by 200 ppm of glutaraldehyde (MBC50 50 ppm) and six strains, including four A. hydrophila, exhibited MBCs of 400–1600 ppm. Glutaraldehyde solutions are commonly used for decontamination of medical equipment. In a recent study, glutaraldehyde phenate failed to prevent A. hydrophila colonization of endoscopes, whilst glutaraldehyde at 2% proved to be efficacious.10 The three species showed a similar susceptibility to free chlorine and cetylpyridinium chloride. However, A. hydrophila was slightly more resistant than A. sobria and A. caviae to glutaraldehyde and peracetic acid, as noticed with chlorine by some investigators.6 There was no correlation between high MBCs of antiseptics and/or disinfectants and antibiotic resistances. In conclusion, the development of drug resistance in European environmental Aeromonas spp. is of clinical concern, both because this is most probably the consequence of the increasing and often indiscriminate use of antibiotics, and because these organisms may cause human infections. The unexpectedly high quinolone resistance rate is particularly worrying, since these antibiotics are first-line drugs against Aeromonas-induced infections.1 The incidence of Aeromonas spp. in chlorinated drinking water and food should be surveyed, in relation to Aeromonas-associated enterocolitis. Further trials using peracetic acid, cetylpyridinium chloride and glutaraldehyde, in various concentrations, formulations and methods of application, are needed to assess the clinical relevance of our in vitro results.
Acknowledgement
1
6 40 17 1 6 54
17 50 6
1 61 29 2
71 26 2 4
100 50 25 12.5 6.25 3.1 1.57 0.78
This work was supported by a PhD grant to M. G. from Navarra Regional Council.
138 138 138 138
References 1. Jones, B. L. & Wilcox, M. H. (1995). Aeromonas infections and their treatment. Journal of Antimicrobial Chemotherapy 35, 453–61. 2. Chang, B. J. & Bolton, S. M. (1987). Plasmids and resistance to antimicrobial agents in Aeromonas sobria and Aeromonas hydrophila clinical isolates. Antimicrobial Agents and Chemotherapy 31, 1281–2.
Glutaraldehyde Chlorine Cetylpyridinium chloride Peracetic acid
0.4 0.2 No. of strains Antiseptic and/or disinfectant
Number of strains with a MBC (ppm) of:
Table II. Antiseptic and/or disinfectant susceptibility of riverine Aeromonas strains
200
400
800
1600
M. Goñi-Urriza et al.
3. Ko, W. C., Yu, K. W., Liu, C. Y., Huang, C. T., Leu, H. S. & Chuang, Y. C. (1996). Increasing antibiotic resistance in clinical isolates of Aeromonas strains in Taiwan. Antimicrobial Agents and Chemotherapy 40, 1260–2. 4. Morita, K., Watanabe, N., Kurata, S. & Kanamori, M. (1994). Beta-lactam resistance of motile Aeromonas isolates from clinical and environmental sources. Antimicrobial Agents and Chemotherapy 38, 353–5. 5. Montoya, R., Dominguez, M., Gonzalez, C., Mondaca, M. A. & Zemelman, R. (1992). Susceptibility to antimicrobial agents and plasmid carrying in Aeromonas hydrophila isolated from two estuarine systems. Microbios 69, 181–6.
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Antimicrobial resistance of river strains of Aeromonas spp. 6. Sisti, M., Albano, A. & Brandi, G. (1998). Bactericidal effect of chlorine on motile Aeromonas spp. in drinking water supplies and influence of temperature on disinfection efficacy. Letters in Applied Microbiology 26, 347–51.
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7. Comité de l’Antibiogramme de la Société Française de Microbiologie. (1999). Communiqué 1999, 1–31. Société Française de Microbiologie, Paris.
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8. AFNOR NF T 72-150 (1988). Méthode par dilution-neutralisation. Recueil de normes françaises. 2nd edn. Association Française de Normalisation, Paris.
Received 3 November 1999; returned 11 January 2000; revised 13 April 2000; accepted 3 May 2000
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